This study investigated the efficacy of first-generation (cefazolin) and third-generation (ceftizoxime) prophylactic antibiotics in patients undergoing cardiac surgery and the incidence of surgical site infections, hospitalizations, and medical costs.All adult patients (≥ 20 years) undergoing cardiac surgery at one hospital from January 01, 2009, to December 31, 2016, were included in this study. A single prophylactic antibiotic was administered at a dose of 1 g within one hour of the surgical incision and for three days after surgery at eight-hour intervals. After propensity score matching, 194 patients in each antibiotic prophylaxis group (first-generation vs third-generation) were analyzed. Among the 388 patients, the incidence of surgical site infections was compared according to the type of prophylactic antibiotic, and risk factors were evaluated by chi-squared tests followed by multivariate logistic regression analysis.The incidence of deep surgical site infections was significantly lower in the first-generation group (5.7%) than in the third-generation group (16.5%). The pathogens isolated from the surgical infection sites were similarly distributed in both groups. However, the prevalence of highly infectious gram-positive bacteria was more than that of gram-negative bacteria (67% vs 23%). The preoperative hospitalization duration, mean operation time, and ventilator use time were similar in both groups, but the postoperative hospitalization duration was significantly shorter in the first-generation group (25.5 days) than in the third-generation group (29.8 days). In addition, the medical costs were lower in the first-generation group  than in the third-generation group .In conclusion, the first-generation prophylactic antibiotic was better than the third-generation in reducing surgical site infection rates, hospitalization length, and medical expenditures.The online version contains supplementary material available at 10.1186/s13019-022-01763-4. In a placebo-controlled trial, the placebo group showed an increased incidence of surgical site infections by 20–50%, confirming the appropriateness of using prophylactic antibiotics in cardiac surgery –3. SurgiStaphylococcus sp. and can be covered by gram-positive bacteria and provides safe and cost-effective treatment but fails to reflect the domestic medical environment because it refers to guidelines from foreign clinical studies. In recent studies, sufficient medical institutions and research subjects were not available, which limited the generalizability of research results [The results of the 2006 Survey on Antibiotic Usage from the National Health Insurance Review and Assessment Service (HIRA) showed that the use of prophylactic antibiotics in Korean surgeries differed from the Guideline for Guidance and has been classified as an abuse of antibiotics , 8. The  results , 10. SinThird-generation prophylactic antibiotic, which was used from 2009 to 2012, was changed to first-generation prophylactic antibiotics due to the evaluation of prophylactic antibiotic use. The use of prophylactic antibiotics has been evaluated since 2012 to promote the prevention of SSIs. The benefit of university hospital institution in changing prophylactic antibiotics has not been identified. Therefore, we performed a comparative study of cephalosporin first-generation (cefazolin) and third-generation (ceftizoxime) antibiotics. The purpose of this study was to investigate the use of prophylactic antibiotics and the prevention of SSIs by analyzing the relationships between the use of prophylactic antibiotics and SSI rates.This study was a retrospective review of the electronic medical records of all patients who underwent cardiac surgery from January 01, 2009, to December 31, 2016, at a single university hospital. All patients had undergone full median sternotomy and cardiopulmonary bypass (CPB). The inclusion criteria were coronary artery bypass grafting (CABG) and valve surgery. Patients with current active infections; those for whom antibiotics had been administered within two weeks of surgery; immunotherapy patients; patients with congenital heart disease, cardiac assistive devices, or extracorporeal membrane oxygenation (ECMO); and patients who had undergone aortic dissection or thoracotomy surgeries were excluded from the study. Of the 554 patients who underwent cardiac surgery, 243 received third-generation antibiotics (ceftizoxime) and 311 received first-generation antibiotics (cefazolin). According to the exclusion criterion, 27 patients in the third-generation group and 45 patients in the first-generation group were excluded. The final study included 216 patients in the third-generation group and 266 in the first-generation group. Following propensity score matching (PSM), a total of 194 patients were categorized into two groups  again. All operations were performed by two experienced cardiac surgeons. The surgical procedures have not been changed to date.The sternal incision sites were evaluated daily by cardiac surgeons and four times a week by an infection management nurse. The diagnosis of identified SSIs was based on positive cultures, dehiscence of the sternotomy, high fever, local pain, redness, purulent drainage, and sternal instability. The duration of the SSI assessments was within 30 days from the beginning of the follow-up period to the end of follow-up based on the SSI guidelines from the CDC . NosocomThe total hospital charges included all medical costs covered by health insurance, self-pay, optional care, and other medical costs. To investigate the costs related to SSIs, the pre-cardiac surgery examination costs, imaging costs, nursing costs, surgical costs, material costs, and admission fees were excluded from the total medical cost. All admission fees were reimbursed based on the admission fee for a six-person room. The hospital charges included post-cardiac surgery medication and treatment costs, examination fees, radiology fees, and treatment material costs for the management of early postoperative complications that occurred during hospitalization.The daily weighted average costs for prophylactic antibiotics were 5.16 US dollars (USD)–5.20 US dollars (USD) for third-generation (ceftizoxime) and 1.08 USD–1.24 USD for first-generation (cefazolin) antibiotics. The exchange rate was based on the rate for November 20, 2019 (1 USD = 1175 Korean won {KRW}).The continuous variables and categorical variables were analyzed by t-tests and chi-squared tests. PSM was used to control selection bias in group selection. This matching method is designed to compare the individual characteristics of two groups based on propensity scores and conditional probabilities. The first-generation and third-generation groups were matched at a 1:1 ratio using the Greedy matching method . Greedy All outcome variables were compared between the propensity score-matched first-generation and third-generation groups by t-tests for numerical variables and chi-squared tests for categorical variables to determine the effect of SSI. The crude and adjusted odds ratios with 95% confidence intervals (CI) were calculated to investigate the independent effect of prophylactic antibiotics on SSI parameters using logistic regression. Incremental cost-effectiveness ratios were calculated for significantly different variables between the two groups. All data analyses were performed using the statistical program SAS version 9.4 .The present study protocol was reviewed and approved by the Institutional Review Board of Inje University Sanggye Paik Hospital . Informed consent was obtained from all patients before enrollment. Before the start of the study, the hospital research ethics review committee received the review. Related laws and regulations were followed throughout the research period.p = 0.028), obesity (p = 0.024), hypertension (p = 0.042), and EuroSCORE risk assessment scores (p < 0.001) were significantly different between the two groups, but there were no statistically significant differences after PSM indicating that matching was balanced. The Hosmer–Lemeshow model goodness-of-fit test statistics were high  . However, in deep SSIs rates in the first-generation antibiotic group (5.7%) was significantly lower than that in the third-generation antibiotic group (16.5%) (p < 0.001). In multiple analysis, after adjusting for the variables of sex, age, diabetes, obesity, smoking, emergency, internal thoracic artery(ITA) use, ventilator, year and ICU stay, the incidence of deep SSIs was significantly lower in the first-generation than in the third-generation group   were significantly less frequent in patients who received first-generation antibiotics (11 of 194 patients (5.6%) than those who received third-generation antibiotics (24 of 194 patients (12.4%), p < 0.01). Also, methicillin-susceptible S. aureus and coagulase-negative Staphylococci were significantly less frequent in the first-generation group (9 of 194 patients (4.4%) than in the third-generation group (25 of 194 patients (7.7%), p = 0.028) , and 1.2 ± 2.2 days for the third-generation and 1.2 ± 2.1 days for the first-generation (p = 0.679), respectively. However, a significant difference was found in the intensive care unit (ICU) stay duration, with 4.1 ± 3.8 days for the third-generation group and 2.9 ± 2.7 days for the first-generation group (p = 0.008). The total hospitalization duration was increased significantly in the third-generation group to 29.8 ± 18.7 days compared to 25.5 ± 20.1 days in the first-generation group (p = 0.025)  and the total hospitalization expenses (p < 0.001) were increased significantly in the third-generation group. The medical costs for non-infected patients were not statistically different (p = 0.092) between two groups but statistical differences for SSIs infected patients were observed in medical costs between the first and third-generation antibiotic groups (p < 0.05). The results showed that medical costs were reduced in the first-generation group, at 5894 USD  and the mean ICU stay duration was 7.3 ± 4.8 days versus 2.4 ± 1.4 days (p < 0.001), respectively, significantly higher in the infected groups. This result may reflect the increased susceptibility to SSIs with the long-term use of ventilators and increased ICU stay duration, leading to increased treatment due to infection. Lola et al. [The duration of hospital stays in the first-generation group was significantly shorter than in the third-generation group. The preoperative hospital length of stay, operating time, and duration of ventilator use did not differ between the two groups. However, in the first-generation group, the ICU stay and hospitalization duration were both significantly shorter than in the third-generation group. In addition, in a comparison of the length of ICU stay between the SSI groups (n = 82) and the non-infected group (n = 306), the mean duration of ventilator use was 2.5 ± 3.7 days versus 0.99 ± 1.4 days (a et al.  reportedProphylactic antibiotic prices vary slightly from manufacturer to manufacturer, but the first-generation is the oldest drug in the classification of cephalosporins and has the lowest cost. The costs of prophylactic antibiotics may be reflected in the overall cost of patient care and treatment. The total medical care expenditure was about 5800 USD higher in the third-generation group, excluding pre-surgery examination fees, cardiac surgery costs, and material costs for the treatment. In particular, while no difference was observed in total medical expenditures between patients in the non-infected group, a significant difference was found in the patients with SSIs. Third-generation antibiotic prophylaxis affected the length of hospitalization and increased the cost of medical care. This was reflected in increases in the SSI rate, hospitalization duration, and medical expenditures for additional treatments . In addiThis study had several limitations as a prophylactic antibiotic study. First, the SSI rate was higher than that of a previous study . The patThe results of this study showed that the use of third-generation prophylactic antibiotics increased the surgical site infection rate and the length of hospital stay compared to the use of first-generation antibiotics. In addition, the microbial cultures showed that the numbers of gram-positive bacteria and antibiotic resistant organisms at the surgical site were high. It is, therefore, important to select suitable prophylactic antibiotics. The selection of first-generation prophylactic antibiotics, with their long-term safety and low cost, was effective in reducing the rate of surgical site infections and decreasing hospitalization and medical expenditures.Additional file 1: Table S1. Microorganisms isolated according to surgical site infections and prophylactic antibiotics.Additional file 2: Table S2. Clinical outcomes of surgical site infections in two-year intervals.


Due to its excellent characteristics, zeolitic imidazole framework-L (ZIF-L) is widely used in various applications, such as drug delivery, wastewater treatments and energy storage. In the synthesis of ZIF-L, the molar ratio of ligand to metal, the reaction time and the temperature are essential parameters to produce excellent ZIF-L. In this work, ZIF-L was synthesized using a facile and green synthesis method. It was statistically investigated and optimized to obtain the best operating conditions. The optimization was carried out toward the amount of adsorbed crystal violet (CV) dye ( Metal–organic frameworks (MOFs) are advanced porous inorganic–organic materials assembled from metal clusters and organic ligands through coordination bonds ,2,3. TheZeolitic imidazolate framework-8 (ZIF-8) is a kind of ZIF that is assembled by coordinating zinc (Zn) as the metal-core and 2-methylimidazole (Hmim) as the organic ligand in the 3D structure. Previous studies have been carried out to synthesise ZIFs, especially ZIF-8, in which various surface areas, pore volumes and pore sizes were obtained ,25,26. ZHerein, a facile, green and optimal synthesis of ZIF-L was carried out in the aqueous system. This synthesis process only used water as the solvent, which was easier to remove and safer for further applications. The molar ratio of ligand to metal, reaction time and temperature were varied in order to obtain the optimum operating conditions of ZIF-L synthesis. The effects of the parameters used were studied using the one-factor-at-a-time (OFAT) method, while the optimization study was statistically modelled using response surface methodology (RSM). Furthermore, the amount of crystal violet (CV) dye adsorbed onto the synthesized ZIF-L  was designed using five levels for each parameter, as presented in F-values and p-values are presented to indicate the significances in p-value of the model term was significant , where this means that the model used fitted the experimental data well. The significances of three independent parameters (linear) and their quadratic and two-way interactions are indicated by an F-value higher than its minimum limit and a p-value lower than 5% (0.05). As shown in F-value and a p-value higher than 0.05. This interpretation is also expressed in the Pareto chart 2.6H2O), 2-methylimidazole (Hmim) and crystal violet dye (CV) were purchased from Sigma-Aldrich (Singapore). All of the chemicals were analytical grade, and were used without further treatment.The zinc nitrate hexahydrate (Zn(NO3)2.6H2O as the metal source and Hmim as the ligand. A metal solution (0.075 M) was prepared in 40 mL distilled water. Another solution was prepared separately containing a specific ligand concentration in another 40 mL distilled water, in which the molar ratio of ligand to metal was varied by 5, 7.5 and 10. Both solutions were sonicated for 10 min, such that the compounds were be well ionized in the solvent. The metal solution was added dropwise with the ligand solution under stirring. The mixture was kept at various temperatures  for various reaction times . In the meantime, the cloudy solution was obtained and then centrifuged for 10 min to separate the ZIF-L precipitation. The precipitation was washed with distilled water and ethanol (twice each), respectively, and dried overnight at 80 °C.The ZIF-L was synthesized from Znq=[ of the response surface methodology (RSM) using Minitab 19 statistical software was applied to design the synthesis of ZIF-L involving cube, axial and center points. There were three independent parameters used, i.e., the molar ratio of ligand to metal .RSM was employed in order to optimize the synthesis of ZIF-L statistically. Herein, the optimum condition was a synthesis condition that produced ZIF-L with the highest adsorption capacity towards crystal violet dye. The linear and quadratic effects of the independent parameters are mathematically expressed by the following equation ,43:(3)Y=1 radiation (λ = 1.5406 Å). It was conducted at 40 kV and 30 mA using a step size of 0.02°/step. Scanning electron microscopy (SEM) was performed using a JSM-6390 field emission SEM, JEOL, Ltd., Japan, at an accelerating voltage of 10 kV and a working distance of 7.5 mm. The samples were prepared using a JFC-1200 coater, JEOL, Ltd., Japan, in an argon atmosphere. A Fourier transform infrared spectrophotometer (FTIR) was used to depict the spectra, indicating the samples’ functional groups. This analysis was carried out using an FTIR Shimadzu 8400S with the KBr pelleting method at a wavenumber range of 4000–400 cm−1. Thermalgravimetric analysis (TGA) was conducted using a Perkin Elmer Diamond TG/DTA thermal analyzer. It was carried out in a nitrogen gas flow of 150 mL/min at a constant heating and cooling rate in a temperature range of 27.5–600 °C.An X-ray diffraction (XRD) pattern was obtained by a PANalytical X’Pert Pro X-ray diffractometer with Cu KαThe synthesis of ZIF-L was successfully carried out under the optimum conditions obtained from the optimization analysis. Statistically, the CCD in RSM was well fitted with the synthesis data, where the optimum condition was the synthesis process using a molar ratio of ligand to metal of 8.2220 for 97 min at 29 °C. This synthesis process is economically promising to produce high-quality ZIF-L, where the molar ratio used was lower than that in previous studies. The ZIF-L produced had a homogeneous starfruit-like shape with excellent crystallinity and thermal stability. It is a promising advanced material that can be used in various applications.


P = 0.002; ΔPANSS positive score, P = 0.01). Plasma cf-mtDNA may represent a relevant tool in the future to assist in the assessment of clinical progression in first-episode patients with schizophrenia.Cell-free mitochondrial DNA (cf-mtDNA) is a damage-associated molecular pattern that boosts the release of cytokines and induces the immune response of the body; therefore, it is closely related to mental diseases. This study aims to evaluate a potential link between cf-mtDNA and clinical progression in first-episode patients with schizophrenia. In this study, plasma cf-mtDNA levels in 34 first-episode patients with schizophrenia before and after 8 weeks of antipsychotic treatment were examined. In addition, the clinical progression of first-episode schizophrenia was assessed using the Positive and Negative Syndrome Scale (PANSS). The copy number changes in the plasma cf-mtDNA (Δcf-mtDNA) were significantly correlated with changes in the PANSS scale scores (ΔPANSS) in first-episode patients with schizophrenia (ΔPANSS total score,  Schizophrenia causes great suffering in afflicted patients, induces a severe burden to affected families, yields a high medical expenditure, and has gradually become a significant social problem . The diaExcess oxidative stress and immune status disequilibrium could be involved in the pathophysiology and represent an underlying mechanism that explains different clinical symptoms of schizophrenia –4. AlthoIn summary, cell-free mitochondrial DNA (cf-mtDNA) reflects both the degree of the oxidative stress condition and immune homeostasis. Furthermore, compared with intracellular mtDNA, cf-mtDNA is easier to obtain, which eliminates complex cell selection operations and facilitates the stability of detection.This study aims to investigate the plasma cf-mtDNA levels in drug-free first-episode patients with schizophrenia. The putative changes in the plasma cf-mtDNA levels during first-episode patients with schizophrenia are also studied using antipsychotic treatments.N = 34) who were diagnosed with first-episode schizophrenia as assessed by the Diagnostic and Statistical Manual of Mental Disorders, 5th Edition (DSM-5) were recruited from Ningbo Kangning Hospital. The subjects had not taken antipsychotics, antidepressants, antimanic, or antiepileptics for at least 2 weeks prior to enrollment. The initial clinical dose of aripiprazole was 10 mg/d, and the dose was gradually increased to a steady concentration of 20 mg/d in the first-episode patients with schizophrenia. Healthy controls (HCs) (N = 28) were matched for age, sex, and body mass index (BMI) to the first-episode patients with schizophrenia. The healthy control participants also met the following criteria: (a) no history of neuropsychiatric disorders; (b) did not meet any of the DSM-5 criteria for the diagnosis of mental disease; (c) no prescription medications  for at least 2 weeks. Furthermore, any subjects with physical conditions, including severe or secondary somatic disease  or a history of smoking, alcohol consumption, or drug abuse, were excluded from this study. Female patients who were pregnant or lactating were also excluded. For all of the subjects, a questionnaire survey was conducted by trained research staff to collect general information, demographic characteristics, and medical conditions. Subjects were excluded if confounding factors  were found in the survey data using the Social Readjustment Rating Scale (score > 150).This study was approved by the Ethics Committee of the Ningbo Kangning Hospital. In the case group, inpatients (MT-ND1) gene was used as the target gene for cf-mtDNA copy number detection, and the hemoglobin (HGB) gene was used as the reference gene. Specific primer sequences were designed using Primer Premier 5.0 software. The MT-ND1 gene was amplified and quantified using the following primers: 5′-CCCTAAAACCCGCCACATCT-3′ (forward) and 5′-GAGCGATGGTGAGAGCTAAGGT-3′ (reverse). The HGB gene was amplified and quantified using the following primers: 5′-GCTTCTGACACAACTGTGTTCACTAGC-3′ (forward) and 5′-CACCAACTTCATCCACGTTCACC-3′ (reverse).The Mitochondrial NADH dehydrogenase subunit 1  and stored at −80°C. The circulating cell-free DNA in the plasma was extracted using a Mag-Bind®cfDNA Kit . The PCR thermocycler  parameters included the following: 95°C for 3 min, 35 cycles of 95°C for 30 s, 57°C for 30 s, and 72°C for 30 s; and a final extension at 72°C for 8 min. Each 25 μl of PCR reaction contained 2.5 μl of 10 × PCR Buffer , 18.3 μl of PCR-grade H2O, 0.5 μl of each primer , and 0.5 μl of the sample DNA.Venous blood was collected in a tube containing EDTA-K2O, 0.2 μl of each primer (10 μM) , and 1.0 μl of the sample DNA (centrifuged supernatant of blood samples diluted five times). cf-mtDNA copy numbers were normalized to that of the nDNA control gene, HGB, using PCR products for the standard curve using pMD 18-T Vector transformation technology .The centrifuged supernatant of the blood samples was used as a template for quantitative real-time PCR . The real-time PCR parameters included the following: 95°C for 3 min; and 45 cycles of 95°C for 15 s, 57°C for 20 s, and 72°C for 30 s. Each 10 μl real-time PCR reaction contained 2 × SybrGreen qPCR Master Mix , 3.6 μl of PCR-grade HThe PANSS assessment was conducted in all of the subjects at both enrollments and the end of the eighth week of the study. Here, the PANSS total score (PANSS-T) was composed of the PANSS positive score (PANSS-P), the PANSS negative score (PANSS-N), and the PANSS general psychopathology score (PANSS-G). The PANSS scale was evaluated using two experienced clinicians. The reliability was determined using the internal consistency test (kappa > 0.90).t-test was used to assess before and after drug treatment, and the group t-test was used to evaluate the comparison between the case group and the healthy control group. The Wilcoxon non-parametric test was used for non-normal distribution analyses. The chi-square test was used to assess the gender distribution between the two groups. The influence of gender, age, and BMI on the plasma cf-mtDNA levels was tested using linear regression. A correlation analysis was used to evaluate the relationship between the plasma cf-mtDNA copy number and the PANSS scores. P < 0.05 was considered to be statistically significant.All of the statistical analyses were performed using the SPSS 19.0 . The normal distributions of the year, the BMI, the cf-mtDNA copy number, and the PANSS scores were assessed using the Kolmogorov–Smirnov test. A paired 2 = 0.01, P = 0.97; t = 0.83, P = 0.41; t = 0.39, P = 0.70). The linear regression analysis on all of the samples showed that gender, age, and BMI had no significant effect on the plasma cf-mtDNA levels .The demographic characteristics of all of the subjects are shown in z = −1.4, P = 0.16), and they were reduced by the use of the antipsychotic treatments     . The cor = 0.84) . Interes = 0.01) .In the present study, consistent plasma cf-mtDNA levels were observed between the first-episode patients with schizophrenia and the HCs. After antipsychotic treatment, the plasma cf-mtDNA levels were reduced and related to the PANSS scores.via the primary response pathway for myelination differentiation leads to the release of cytokines, such as interleukin 6 (IL-6) (It was found that first-episode patients with schizophrenia showed similar plasma cf-mtDNA levels to those of the HCs. This result suggested that this parameter had no association with schizophrenic risk. This result was similar to that of the previous study . The res6 (IL-6) , 15. IL-6 (IL-6) , 22. TheThe present study had some limitations. First, the sample size was very small, and the average age of the patients was older than expected. Second, there was a lack of other measurements in addition to the cf-mtDNA . Third, the study  may not be generalized to all patients with schizophrenia. Fourth, the reasons for the cf-mtDNA levels changes remain unclear and require further research.In summary, the results suggested that plasma cf-mtDNA levels were reduced in drug-free first-episode patients with schizophrenia after 8 weeks of antipsychotic treatment, and the change in the PANSS scores was associated with the change in the plasma cf-mtDNA levels. In the future, the use of plasma cf-mtDNA levels may be a relevant tool to assist in the assessment of clinical progression in first-episode patients with schizophrenia.The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author.The studies involving human participants were reviewed and approved by Ethics Committee of Ningbo Kangning Hospital. The patients/participants provided their written informed consent to participate in this study.DZ and HO: conceptualization and validation. HO: methodology and writing the original draft preparation. MH and QY: software. YX: formal analysis. DZ, XW, and MH: resources. DZ: writing the review and editing, and project administration. All authors have read and agreed to the published version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The movement disorders of dyskinesia were detected by the abnormal involuntary movements scores analysis. Effects of RES on glial cells-elicited neuroinflammation were also explored. Data showed that RES attenuated dyskinesia induced by L-DOPA without affecting L-DOPA’s anti-parkinsonian effects. Furthermore, RES generated neuroprotection against long term treatment of L-DOPA-induced DA neuronal damage. Meanwhile, RES reduced protein expression of dyskinesia molecular markers, ΔFOS B and ERK, in the striatum. Also, there was a strong negative correlation between DA system damage and ΔFOS B level in the striatum. In addition, RES inhibited microglia and astroglia activation in substantia nigra and subsequent inflammatory responses in the striatum during L-DOPA treatment. RES alleviates dyskinesia induced by L-DOPA and these beneficial effects are closely associated with protection against DA neuronal damage and inhibition of glial cells-mediated neuroinflammatory reactions.Dyskinesia is a serious complication of Parkinson’s disease during levodopa (L-DOPA) treatment. The pathophysiology of L-DOPA-induced dyskinesia (LID) is complex and not fully illuminated. At present, treatment of dyskinesia is quite limited. Recent studies demonstrated neuroinflammation plays an important role in development of LID. Thus, inhibition of neuroinflammation might open a new avenue for LID treatment. Resveratrol (RES) is the most well-known polyphenolic stilbenoid and verified to possess a large variety of biological activities. DA neurotoxicity was assessed  Parkinson’s disease (PD) is the second most prevalent neurodegenerative disease in the world with an incidence of about 1%-2% . Patientvia the decreased IL-1β production in the striatum  glutamate receptor. However, it could not be an ideal drug for long-term treatment of dyskinesia due to its tolerance and side effects . To findvia the activation of SIRT1 signaling  is the most well-known polyphenolic stilbenoid, found in grapes, peanuts, mulberries and several other plants . RES hasignaling . Althougignaling .Currently, DA neuronal loss is one of the pathological features of PD, which has been recognized to be an important risk factor for the development of LID . Based oMale Sprague-Dawley rats weighing 180-200 g were used. Rats were housed in a temperature (19-25°C) and humidity-controlled (40-70%) with a 12 h light/dark cycle and free access to autoclaved water and rat chow diet. All efforts were made to minimize their suffering. A total of 160 rats were randomly divided into 4 groups with 40 rats in each group, namely: 6-OHDA, 6-OHDA+RES, 6-OHDA+L-DOPA and 6-OHDA+L-DOPA +RES groups. Then, ten rats were sacrificed at each time point, in which 5 rats were applied for immunohistochemical staining and the other 5 rats were used for western blot analysis. All animal experiments were performed in accordance with National Institute of Health Guideline for the Animal Care and Use of Laboratory Animal and the protocols were approved by the institutional Animal Care and Use Committee at Zunyi Medical University .To create the 6-OHDA lesion, rats were anesthetized with sodium pentobarbital (60 mg/kg) and then immobilized in a stereotaxic frame to target the unilateral SNpc  relative to bregma. A single 6-OHDA  solution was injected over 3 min with an infusion rate of 1 μL/min, followed by 3 min of equilibrium before retracting the needle. All rats were allowed to a 21-day recovery period before experiment start. Then, intraperitoneal injection of L-DOPA  and intragastrical administration of RES  were performed daily.The rotarod treadmills were made of a rotating spindle (7.6 cm diameter) and 4 individual compartments able to simultaneously test four rats. Before the experiment, rats were subjected to two days of adaptive training, and the speed was fixed at 12 rpm. In the formal test, the acceleration parameter was selected to be 4.0-40 rpm for the experiment. Each rat was tested 3 times with an interval of 30 min. Finally, the average stay time of 3 times in the rod of each rat was statistically analyzed.The stepping test was employed to measure rat forelimb akinesia. Rats with unilateral DA depletion of more than 80% performed poorly in this test . L-DOPA L-DOPA induced AIMs scores were evaluated on 7, 21, 42 and 84 days after L-DOPA treatment. Dyskinesia behavior changes were assessed for 1min every 30-min interval over a 120-min period. The following three subtypes of AIMs: Axial, Limb and Orofacial AIMs scores were evaluated. Each subtype AIM was scored based on a frequency from 0 to 4 . Through the AIMs test, the severity of dyskinesia in each rat was quantified. The total AIM scores were obtained by summing each AIMs subtype score from each 30-min observation period.After animals were deeply anesthetized, brains were quickly dissected and separated on ice and the separated midbrain and striatum tissues were frozen at -80°C. The frozen tissue was homogenized with lysis buffer (including protease inhibitor and phosphate protease inhibitor) and cracked on ice. The supernatant was collected after centrifugation for 10 min at 4°C. Protein concentrations were detected by BCA assay kit. An equal of amount of protein for each sample was loaded onto an sodium dodecyl sulfate polyacrylamide electrophoresis gel (10%). Proteins separated by gel electrophoresis were electro-transferred onto a 0.45 mm polvinylidene difluoride membranes. The membranes were blocked with 5% skimmed milk, and then incubated with the following primary antibodies at 4°C overnight: rabbit anti-TH , rabbit anti-IL-1β , rabbit anti-TNF-α , rabbit anti-COX-2 , rabbit anti-Inos , rabbit anti-p-ERK (Thr202/204) , rabbit anti-ERK , rabbit anti-ΔFOS B , rabbit anti-GAPDH , rabbit anti-β-actin . After washing, bound antibodies were detected with HRP-conjugated secondary anti-rabbit antibody . The blots were developed with enhanced chemiluminescence antoradiography (ECL kit) and quantified with the software Quantity one.via high temperature and pressure antigen repair using citrate buffer (pH=6). Tissue was cooled to room temperature and incubated in goat serum for 30 min, and incubated with the following primary antibodies at 4 overnight: rabbit anti-TH , rabbit anti-IBA-1 , rabbit anti-GFAP , rabbit anti-ΔFOS B . For immunohistochemical staining, slides were incubated with biotinylated secondary antibody at 37°C for 15 min and visualized with avidin horseradish enzyme at 37°C for 20 min. Slices were visualized through DAB kit. For immunofluorescence staining, the second antibodies were anti-rabbit IgG Alexa Flour 488 . Quantification of TH-positive neurons was performed through visually counting the number of TH-positive neuronal cell bodies blindly by two investigators. The images were recorded with a charge-coupled device camera and operated with the MetaMorph software. The results were obtained from the average. The mean value for the number of TH-positive neurons in substantia nigra was then deduced by averaging the counts of six sections for each rat.Rats were perfused intracardially with PBS and subsequently fixed with 4% paraformaldehyde. After tissue was dehydrated and embedded in paraffin, a total of 5-μm section was cut and collected. Antigens were repaired post hoc t-test with correction. A value of p<0.05 was considered statistically significant. In addition, the correlation between DA system damage and ΔFOS B level was tested by Pearson correlation analysis.Data were expressed as mean ± standard error of the mean (SEM). Statistical significance was analyzed by one- or two-way analysis of variance (ANOVA) using GraphPad Prism software . When ANOVA showed significant difference, pairwise comparisons between means were analyzed by Bonferroni’s Rat Dyskinesia behavior dysfunction induced by L-DOPA was evaluated by AIM scores 7, 21, 42 and 84 days after L-DOPA application. As shown in L-DOPA’s efficacy of anti-PD motor dysfunction was assessed by rotarod test. As shown in Next, the effects of RES combined with L-DOPA on 6-OHDA-induced DA neuronal loss were investigated. As shown in After 42 days of L-DOPA administration, the effects of L-DOPA or RES on protein expression of ΔFOS B, phosphorylated-ERK (p-ERK) and total ERK in the ipsilateral striatum of rats were detected. As shown in Since neuroinflammation was considered to be one of the important causes of LID, the role of glial cells-mediated neuroinflammation in RES-attenuated LID was explored. As shown in The purpose of this study was to determine whether the “multi-target drug” RES alleviated dyskinesia induced by L-DOPA. Results clearly indicated RES attenuated LID without affecting L-DOPA’s anti-parkinsonian effects. Furthermore, RES generated neuroprotection against long term treatment of L-DOPA-induced DA neuronal damage. Meanwhile, RES reduced the protein expression of dyskinesia molecular markers, ΔFOS B and ERK, in the striatum. Also, there was a strong negative correlation between DA system damage and ΔFOS B level in the striatum. In addition, RES inhibited microglia and astroglia activation in substantia nigra and subsequent inflammatory responses in the striatum during L-DOPA-treated PD. Collectively, these results suggested that RES attenuated LID through protection against DA neuronal damage and inhibition of glial cells-mediated neuroinflammatory reactions.Nowadays, clinical studies indicated that the course of PD was a risk factor for the onset of dyskinesia , and theAt present, the pathogenesis of LID is very complicated. As mentioned earlier, the decrease of DA projecting fibers in the striatum of PD led to rapid changes in the concentration of DA neurotransmitters after L-DOPA treatment, resulting in changes in the conduction function of basal ganglia. A number of studies showed that the increased activity of D1R led to changes in a series of downstream molecular levels, such as phosphorylation of cyclic adenosine monophosphate phosphorylated protein-32 (DARPP-32) , ERK1/2 Although neuroinflammation is verified to contribute to the pathogenesis of PD, recent studies have confirmed that neuroinflammation also plays an important role in LID . The rolTaken together, this study revealed the role of anti-inflammatory therapy on improving the occurrence of dyskinesia upon L-DOPA treatment. Results demonstrated that RES combined with L-DOPA could reduce further loss of DA neurons and attenuated the severity of LID. However, this study only focuses on the therapeutic effect of RES on dyskinesia induced by L-DOPA. Next, to further investigate whether anti-dyskinetic effects of RES are reversible after withdrawal or prior to L-DOPA warrants future investigation .RES alleviated dyskinesia induced by L-DOPA and these beneficial effects were closely associated with protection of DA neuronal damage and inhibition of glial cells-mediated neuroinflammatory responses.The original contributions presented in the study are included in the article/All animal experiments were performed in accordance with National Institute of Health Guideline for the Animal Care and Use of Laboratory Animal and the protocols were approved by the institutional Animal Care and Use Committee at Zunyi Medical University .C-QZ participated in the design, execution, data analysis and writing of the manuscript. H-XF, X-XL, J-JL, and SS participated in the revision of the article, and FZ participated in the design and revision of the article. All authors contributed to the article and approved the submitted version.This study was supported by the National Natural Science Foundation of China (No. 81760658), the foundation for High-level Innovative Talents of Guizhou Province (No. 20164027), the Innovation Research Group project of Education Department of Guizhou Province (No. 2016038) and the foundation for Excellent Young Talents of Zunyi Medical University (No.201603).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Introduction: Coronavirus disease 2019 (COVID-19) vaccines protect against severe illness. However, data on post-vaccination COVID-19 breakthrough infections are limited.Methods: An analytical cross-sectional study was conducted from May 2021 to July 2021 among 2043 COVID-19-positive healthcare workers who were divided into a vaccinated group (n=1010) and an unvaccinated group (n=1033). A pre-tested questionnaire was circulated among the healthcare workers using Google Forms. Chest computed tomography (CT) severity score was the primary outcome variable analyzed using coGuide.p = 0.07 and p <0.001, respectively). However, the difference was insignificant (p >0.05) for gender, hypertension, and diabetes. Most unvaccinated respondents had an increased CT severity score, and the difference between the studies groups was significant (p <0.001). Of the 1,010 vaccinated individuals, 382 (37.82%) received the only first vaccination dose, and 628 (62.18%) received both doses. The CT severity score decreased after receiving both vaccination doses, and the difference between CT severity score and vaccination status was significant (p <0.001).Results: The average age of the study population was less than 45 years in both groups (43.05 ± 13.02 years). Most respondents (62%) were males. Hypertension (39%) and diabetes (33%) were the most common underlying diseases. Significant differences in age and cardiac disease were observed between the two groups (Conclusion: COVID-19 was mild in the vaccinated group. Chest CT severity score index can be considered an efficient tool in predicting prognosis and monitoring disease in patients with COVID-19 in India. Since its emergence in December 2019, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has caused high morbidity and mortality, with new variants rapidly spreading. Vaccines have been developed to prevent coronavirus disease 2019 (COVID-19) with unprecedented speed [The first vaccines against SARS-CoV-2 have now received emergency use authorization by the US Food and Drug Administration, European Medicines Agency, and national authorities in China, India, the UK, and Russia and are rapidly being distributed in more than 30 countries since the end of 2020 [Real-world studies from Israel and England have reported 92% , 88% to 95%) effectiveness against symptomatic polymerase chain reaction (PCR)-confirmed infection ≥seven days after the second Pfizer-BioNTech dose ,5. RecenThough COVID-19 vaccines protect against severe illness, post-vaccination SARS-CoV-2 infections are expected, as 100% protection is not offered by these vaccines (breakthrough infections) . The curStudy designAn analytical cross-sectional study was conducted using an online survey among 2,043 healthcare workers of Karnataka from May 2021 to July 2021. Ethical permission was obtained from the Institutional Review Board of the concerned tertiary care center (reference number: SDUMC/KLR/IEC/243/2021-22). Written informed consent was obtained from all participants. The information present in the questionnaire was explained to the participants in detail.Sample size calculationThe sample size was calculated assuming that the proportion of patients with severe COVID-19 with chest CT severity score was 16.7% in the vaccinated group and 29.9% in the unvaccinated group according to the pilot study. The other parameters considered for sample size calculation were 90% power and 95% confidence level. The required sample size according to the aforementioned calculation was 210 in each group. To account for a nonparticipation rate of approximately 30%, another 63 subjects were added to the sample size. Hence, the final required sample size would be 273 in each group. According to the availability of cases, 1,010 and 1,033 in the vaccinated and unvaccinated groups, respectively, were considered as per convenience sampling method for the feasibility of the study.Inclusion criteriaPatients infected with COVID-19 after vaccination who tested positive on RT-PCR and have undergone high-resolution CT (HRCT) of the thorax and patients infected with COVID-19 who were unvaccinated, tested positive on RT-PCR, and have undergone HRCT of the thorax.Exclusion criteriaPatients who have tested positive on RT-PCR and have not undergone HRCT of the thorax and patients who have tested negative on RT-PCR and have undergone HRCT of the thorax.Data collectionThe questionnaire was circulated among healthcare workers of Karnataka using Google Forms. The questions were developed to collect data regarding vaccination status, COVID-19 positivity after vaccination, and CT severity score. Few questions were used from a previous retrospective study on COVID-19-positive healthcare workers post-vaccination by Patil et al. , in MumbThe Dutch Radiological Society created the CO-RADS, which standardizes the assessment scheme and simplifies reporting using a five-point scale of suspicion for COVID-19 pneumonia on chest CT images.The CO-RADS categories used for CT scoring are summarized as follows Table Each patient was categorized as CO-RADS 0, 1, 2, 3, 4, 5, or 6 based on the aforementioned findings.The CT severity score index ,14 is a Study variablesCT severity score was the primary outcome variable.Statistical methodsDescriptive data were presented as means ± standard deviations for age and as frequency and proportions for gender and comorbidities. Age and comorbidities were compared across the groups using an independent t-test, and CT severity scores were compared among the groups using the chi-square test. P-values of less than 0.05 were considered statistically significant. Data were analyzed using coGuide software (V.1.03) .All 2,043 subjects were considered for final analysis.Of the 2,043 healthcare workers, 49.4% received vaccination and 50.6% were unvaccinated. The ratio of vaccinated and unvaccinated individuals in the study population was almost equal.The mean age was 43.05 ± 13.02 years in the vaccinated group and 42.05 ± 13.15 years in the unvaccinated group. Most respondents (62%) were males in both groups. Hypertension (39%) and diabetes (33%) were the most common underlying diseases in both groups. Moreover, 98 (9.70%) healthcare workers in the vaccinated group had cardiac disease, whereas only 13 (1.26%) healthcare workers in the unvaccinated group had cardiac disease. However, significant differences in age and the prevalence of cardiac disease were observed between the two groups , and the differences in sex ratio and comorbidities, such as hypertension and diabetes, between the two groups were insignificant (p >0.05)  showed severe CT scores, whereas, of the 1,010 individuals in the vaccinated group, only three had severe CT scores. The difference in CT severity score between the study groups was significant (p <0.001)  received only the first dose and 628 (62.18%) received both vaccination doses (Table The CT severity score was reduced after receiving both vaccination doses. Only 10 (1.59%) of the 628 individuals who received both vaccination doses showed moderate severity scores after receiving both vaccination doses compared with those who received only a single dose of vaccination where 101 (26.44%) had moderate scores after receiving the first dose. The difference in CT severity score and vaccination status was significant (p <0.001) Table .To the best of the author’s knowledge, this is the first study that has assessed the severity of COVID-19 using CT severity scores among vaccinated and unvaccinated COVID-19-positive healthcare workers of Karnataka. The CO-RADS classification developed by the Dutch Radiological Society is a simple interpretation method with a high inspection accuracy and matching rate. This is one of the few studies that verified the effectiveness of the CO-RADS in assessing the severity of COVID-19. The difference in chest CT severity scores between the study groups was significant (p <0.001) with severe scores observed only in three (0.3%) healthcare workers in the vaccinated group compared with 154 (14.91%) healthcare workers in the unvaccinated group. As data related to breakthrough infections after vaccination for COVID-19 are limited, we included nearby studies related to COVID-19 to compare the findings of this study. The mean age was 43.05 ± 13.02 years in the vaccinated group and 42.05 ± 13.15 years in the unvaccinated group, and the difference in age was statistically significant (p = 0.07). Moustsen-Helms et al.  found a In the present study, the CT severity score was reduced after receiving both vaccination doses, and the difference between CT severity score and vaccination status was significant (p <0.001). Hitchings , in his Most vaccinated and unvaccinated individuals had diabetes and hypertension as underlying diseases. Sanyaolu et al. , in theiMuruato et al.  have devThis study has a few limitations. First, the study design was cross-sectional, which can affect the causal and temporal relationships. The study was conducted on healthcare workers and hence cannot be generalized to the general population. As the data from healthcare workers was collected online the information provided can be subjected to recall biases. Moreover, some important variables, including information on the type of vaccination, hospitalization due to COVID-19, and duration post-vaccination that can affect the efficacy of vaccination, were not recorded. The relationship between CT severity image findings and disease prognosis was not examined. Further research will be needed to validate the findings of this study and to compare the diagnostic performance of the CO-RADS in assisting radiologists and improving their accuracy and efficiency in diagnosing COVID-19. This was not an efficacy study or a randomized controlled trial, and hence, further multicentre studies with such designs covering a large sample of all age groups are recommended.COVID-19 infection can occur even after vaccination among healthcare workers as they are at the highest risk of exposure. CT severity scores were mild in the vaccination group compared with those in the un-vaccination group. Chest CT must be considered the primary diagnostic tool in predicting disease severity in patients with COVID-19. Global vaccine coverage and infection control practices, such as wearing a mask, social distancing, wearing appropriate personal protective equipment, and hand sanitization, will help combat the pandemic. In the pandemic period, laboratory testing for COVID-19 should be performed to differentiate COVID-19 symptoms from side effects of the vaccine in the vaccinated population. During subsequent waves of this novel virus transmission, vaccination can decrease mortality, and disease severity. The chances of getting COVID-19 are low after vaccination because the vaccine helps protect the body against the virus and in case of re-infection, the vaccine reduces the severity of COVID-19 and the possibility of hospital admission and death.


Since 2020, humanity has been facing the COVID-19 pandemic, a respiratory disease caused by the SARS-CoV-2. The world’s response to pandemic went through the development of diagnostics, vaccines and medicines. Regarding diagnostics, an enormous challenge was faced due to shortage of materials to collect and process the samples, and to perform reliable mass diagnosis by RT-qPCR. In particular, time-consuming and high cost of nucleic acid extraction procedures have hampered the diagnosis; moreover, several steps in the routine for the preparation of the material makes the extracted sample susceptible to contamination. Here two rapid nucleic acid extraction reagents were compared as extraction procedures for SARS-CoV-2 detection in clinical samples by singleplex and multiplex RT-qPCR analysis, using different transport media, samples with high and low viral load, and different PCR machines. As observed, rapid nucleic acid extraction procedures can be applied for reliable diagnosis using a TaqMan-based assay, over multiple platforms. Ultimately, prompt RNA extraction may reduce costs with reagents and plastics, the chances of contamination, and the overall time to diagnosis by RT-qPCR. COVID-19, caused by SARS-CoV-2, became a global pandemic disease from March of 2020, when the World Health Organization (WHO) declared a “public health emergency of international concern” . In DeceThe virus that causes the disease belongs to the Coronaviridae family, which has an RNA genome. Genetic sequencing showed that SARS-CoV-2 is 79% equivalent to SARS-CoV and 50% to MERS-CoV . Thus faThe most used methods for detecting or quantifying viruses nucleic acids in human biological samples are reverse transcription quantitative polymerase chain reaction (RT-qPCR) or real-time PCR. This technique detects a small segment of the viral genome, enabling rapid, sensitive and accurate strain-level detection of up to five targets in an assay. Several RT-qPCR assays have been designed for the detection of SARS-CoV-2 ,11. CurrOne of the bottlenecks to the diagnosis of COVID-19 by RT-qPCR is the limited availability of RNA extraction kits for preparing the samples collected from patients. The extraction is usually time-consuming and becomes a problem due to the urgency required for diagnosis ; moreoveTM DNA Extraction Solution, by Lucigen Corporation . Once thawed, aliquots were stored at −20 °C to avoid >3 freeze–thaw cycles.In this study, two rapid nucleic acid extraction reagents were used: Pi-Lise Nucleic Acid Extraction Reagent, by Pi-Biotech Genética Avançada Ltda , and QuickExtract™ 3 and 7500Fast Real-Time PCR Systems, by Applied Biosystems .To perform the RT-qPCR assays, a multiplex kit , and a singleplex set of reagents . The analyses were run in two distinct equipment: QuantStudioBiological samples for this study were collected from patients suspected of COVID-19 between August 2020 and August 2021. A total of 50 respiratory samples  with known results emitted by local diagnostic laboratories were included in the study. The positive samples were chosen to present both high and low viral load .The study was submitted and approved by the local ethics committee (CEP UFJF—CAAE: 48021321.3.0000.5147) and included written consent from all participants.The method used to perform the extraction of nucleic acids from the samples was based in Ladha et al. , with slFor the extraction, first the biological samples in viral transport medium (VTM) or saline (NaCl 0.9%) were diluted 1:1 with the extraction reagents. Here, a volume of 40 µL of sample was diluted with 40 µL of Pi-Lise or QuickExtract  and homoThe real-time PCR assay was performed using multiplexed and singleplexed approaches: the Allplex™ SARS-CoV-2 Assay , and iTaq Universal Probes One-Step Kit , with primers, probes and controls 2019- nCoV RUO (IDT Integrated DNA Technologies).Allplex™ SARS-CoV-2 Assay is a multiplexed RT-qPCR assay that detects and identifies in a single sample three target: RdRp and N genes (specific for SARS-CoV-2) and gene E  ,17. On tThe real-time PCR was carried out in the 7500 Fast Real-Time (Applied Biosystems) for the multiplex assay. The final 25 μL reaction mixture contained 3.0 µL of extracted sample, 5.0 µL of primers and probes, 7.0 μL of enzyme mix and reaction buffer and 10.0 μL of nuclease-free water. The thermocycling conditions consisted of 50 °C for 20 min for reverse transcription, followed by 95 °C for 15 min and then 45 cycles of 95 °C for 15 s, 58 °C for 30 s.The following reactions were performed using Bio-Rad Laboratories singleplex kit and IDT primers in a QuanStudio3 Real-Time PCR System (Applied Biosystems). The final 20 µL reaction mixture contained 3.0 µL of extracted sample, 0.5 μL of iScript enzyme mix, 10 μL of 2x iTaq PCR reaction mix and the combination of primers/probes and water . Cycling was performed at 50 °C for 15 min for reverse transcription, followed by 95 °C for 3 min and then 40 cycles of 95 °C for 15 s, 60 °C for 1 min .All runs included a positive SARS-CoV-2 genomic control and a negative control for the PCR amplification step. Fluorescence measurements were taken, and the threshold cycle (CT) value was calculated by determining the point at which fluorescence exceeded a defined threshold of the mean plus 10 standard deviations above baseline ,21, accoTo investigate the interference of Pi-Lise reagent on RT-qPCR activity, RT-qPCR reactions using SARS-CoV-2 synthetic gene fragment dissolved in nuclease-free water or in a 50:50 nuclease-free water:Pi-Lise mixture were compared, as described by Ladha et al. for QuickExtract reagent . Each RTp < 0.05 were considered statistically significant. Graphs were prepared using GraphPad Prism version 8.0.1 (USA).Statistical analysis was performed using GraphPad Prism version 8.0.1 (USA). Differences in the mean of cycle threshold (CT) values of genes were compared between the rapid extraction reagents by the T-Student test. Values with Initially, the analyses were performed using the Allplex kit (Seegene), on the 7500 Fast Real-Time. The results presented in p < 0.05). As for the RdRp gene, there was no statistical significant difference between the mean CT values of Pi-Lise and QuickExtract. Ultimately, the obtained results showed that Pi-Lise and QuickExtract were effective in providing nucleic acid from samples in the different media tested. The detection of genes N, E and RdRp was viable in biological samples regardless of the transport media used   .A singleplex analysis  was also performed after the extraction with both reagents. For this analysis, the QuantStudio 3 equipment was used. The results show that there was no significant difference in the final outcome when the samples were processed with Pi-Lise or QuickExtract . The prov/v did not interfere with the RT-qPCR reaction.A series of reagent dilutions were evaluated to identify one that would achieve efficient lysis of the enveloped virus while preserving the activity of the RT-qPCR reaction. The results  show thaThis study compared two commercially available reagents on the extraction of nucleic acid from biological samples for COVID-19 diagnostic by RT-qPCR. The reagents Pi-Lise and QuickExtract promoted rapid extraction of nucleic acids, without the application of columns and organic solvents , for theThe results obtained in this study show that SARS-CoV-2 was identified in different clinical samples after nucleic acid extraction by both reagents used separately. In addition, Pi-Lise and QuickExtract were effective in extracting viral RNA from endotracheal aspirate. These results demonstrate that the reagents are effective in complex matrices.In nasopharyngeal swab samples, transported in viral transport medium and/or saline solution, Pi-Lise and QuickExtract were also efficient for nucleic acid extraction from samples with high viral load (CT ≤ 30.0) and for samples with low viral load (CT ≥ 30.0). In a study by Komiazyk et al., difficulty in extracting RNA from samples with low viral load was reported, in particular, due to operational difficulties in conventional extraction techniques (by column or organic solvents) . Due to Based on the CT values obtained, both reagents showed similar results for gene N. Gene N is part of the viral nucleocapsid and has been reported as a long region of the viral gene sequence, highly conserved among Sarbecoviruses . AccordiFor the elaboration of the E gene primer–probe, a region of the viral structural protein envelope was used . AlthougIn the analysis using a singleplex test, the extraction process employing Pi-Lise or QuickExtract was also satisfactory for the final detection of SARS-CoV-2 in different samples. In these reactions, the panel of primers used was that of the US CDC, which contained the genes N1 and N2, specific to SARS-CoV-2 and the RNAseP gene, as endogenous control . Accordiv/v). In these samples, amplification was not observed in samples without positive control and the virus was detected at all concentrations tested. This result shows that Pi-Lise does not interfere in RT-qPCR reactions.The Pi-Lise interference analysis in the RT-qPCR reaction showed that the reagent does not harm the PCR assay. The reaction volume was 25 µL, with 3 µL of sample. Among the samples containing Pi-Lise:Water mixture (50:50), the largest volume of Pi-Lise analyzed was 1.5 µL , using different equipment and diagnostic sets. Reliable results can be obtained using rapid nucleic acid extraction reagents and methods, with a positive impact on time and cost of analysis.


We aimed to compare cardiovascular outcomes of patients with type 2 diabetes (T2D) who initiated GLP-1 receptor agonists (GLP-1RA) or basal insulin (BI) under routine care.We accessed the administrative claims database of the Veneto Region  to identify new users of GLP-1RA or BI in 2014–2018. Propensity score matching (PSM) was implemented to obtain two cohorts of patients with superimposable characteristics. The primary endpoint was the 3-point major adverse cardiovascular events (3P-MACE). Secondary endpoints included 3P-MACE components, hospitalization for heart failure, revascularizations, and adverse events.From a background population of 5,242,201 citizens, 330,193 were identified as having diabetes. PSM produced two very well matched cohorts of 4063 patients each, who initiated GLP-1RA or BI after an average of 2.5 other diabetes drug classes. Patients were 63-year-old and only 15% had a baseline history of cardiovascular disease. During a median follow-up of 24 months in the intention-to-treat analysis, 3P-MACE occurred less frequently in the GLP-1RA cohort . All secondary cardiovascular endpoints were also significantly in favor of GLP-1RA. Results were confirmed in the as-treated approach and in several stratified analyses. According to the E-value, confounding by unmeasured variables were unlikely to entirely explain between-group differences in cardiovascular outcomes.Patients with T2D who initiated a GLP-1RA experienced far better cardiovascular outcomes than did matched patients who initiated a BI in the same healthcare system. These finding supports prioritization of GLP-1RA as the first injectable regimen for the management of T2D.The online version contains supplementary material available at 10.1186/s12933-021-01414-3. Since its discovery in 1921, insulin has become the mainstay of diabetes management. After 100 years, insulin use is still compulsory for correcting hyperglycemia in individuals with type 1 diabetes. On the other side, pharmaceutical developments have provided several valid alternatives to insulin for the management of type 2 diabetes (T2D). When insulin therapy is required to treat T2D, basal insulin (BI) is the preferred initial approach, because it allows similar glycemic improvement as compared with more intensive regimens, but with lower hypoglycemia risk  recommenRandomized controlled trials (RCTs) have shown that, compared to BI, GLP-1RA granted similar or greater HbA1c reductions, but with lower rates of hypoglycemia, substantial weight loss, and easier administration schedule . In addiIn the absence of evidence from RCTs, real-world studies on hard outcomes have compared several classes of glucose-lowering medications, including GLP-1RA, dipeptidyl-peptidase 4 inhibitors (DPP-4i), and sodium glucose cotransporter-2 inhibitors (SGLT2i). Patients initiating GLP-1RA under free-living conditions had better cardiovascular outcomes compared to those initiating DPP-4i , whereasHere, we wished to verify if, in routine clinical practice, patients with T2D who initiated a GLP-1RA exhibited better cardiovascular outcomes than similar patients who initiated BI at the same disease stage and with the same degree of comorbidity. To this end, we conducted a Region-wide retrospective study on propensity score matched cohorts of patients.This was a Region-wide, retrospective, longitudinal, comparative effectiveness study. The framework for this study has been describe previously , 13, 16.The background population of patients with diabetes mellitus was selected among Italian citizens residing in Veneto who had been eligible beneficiaries for at least 1 year between January 1st, 2011 and September 30th, 2018 or time of death. In order to identify individuals with diabetes, we used an algorithm based on claims data and validated against the gold standard clinical diagnosis [We defined two analytical approaches. In the “ITT” approach, patients were followed from the index date to the event, death, or the last available observation, whichever occurred first. In the “as treated” (AT) approach, the observation was stopped in case patients discontinued treatment with GLP-1RA or BI. Discontinuation was defined as lapse in GLP-1RA or BI prescription longer than 12 months; censoring happened at the end of the 12-month period. The 12-month gap was identified as part of an empirical correlation analysis between discontinuation time as apparent within specialist records  vs. administrative claims (the main data used for this study).The primary outcome for this study was a modified version of the 3-point MACE (3P-MACE), defined as occurrence of myocardial infarction, stroke, or all-cause death. Death from any cause was used in this version of the 3P-MACE in place of death from cardiovascular causes because causes of death were not available in the database. Individual components of the 3P-MACE were secondary endpoints, along with additional outcomes: hospitalization for heart failure (HHF), hospitalization for any cardiovascular disease (CVD), and revascularization procedures. To ascertain outcomes, we used ICD-9-CM codes from hospital discharge claims: myocardial infarction (410–414), stroke (431–436), hospitalization for heart failure (428), hospitalization for any cardiovascular cause (390–459), and revascularization . Time to event was coded in months to comply with the time resolution of anonymized dates of death in the administrative database.We evaluated occurrence of a series of adverse events during the observation, which are of particular interest for GLP-1RA and BI: pancreatic cancer , pancreatitis (577.0), acute and chronic kidney disease (584), diabetic ketoacidosis (250.1), and severe hypoglycemia . Validity of claims-based diagnosis for pancreatic cancer was confirmed in at least three independent databases –21, wherData are presented as mean  for continuous variables, or as percentage for categorical variables. Due to the real-world nature of the study, differences in patients’ characteristics between the two groups were expected. To obtain two subgroups of patients that were comparable, we used propensity score (PS) matching (PSM) with the nearest neighbor method and the logit distance, and a maximum caliper 0.5% SD of PS. To estimate the PS, we used a logistic regression model with the following covariates: age at index date, sex, clinical history in the claim database (months between the first available claim and the index date), claims-based diabetes duration ; history of a first specialist diabetes visit in the preceding year; concomitant risk factors , diabetic complications , history of severe hypoglycemia, other comorbidities , the Charlson comorbidity , thAfter PSM, we used the Mann–Whitney’s U test or the chi-square test or to evaluate the balance in continuous or categorical variables, respectively. The match was considered to be successful when p-values for all comparisons were greater than 0.05 or the differences were small (absolute standardized mean difference < 0.10). This implies that, with large sample size, differences yielding a p-value < 0.05 would be considered non-clinically significant if SMD was < 0.10 (i.e. a difference that is < 10% the pooled SD for continuous variables).The primary analysis was performed using the ITT approach, comparing hazard ratios (HRs) for GLP-1RA versus BI initiators in terms of 3P-MACE, its individual components, and other secondary outcomes. We performed the following secondary analyses: (i) a repeat of the primary analysis using the AT approach; (ii) a comparison of all endpoints in the ITT dataset stratified by the history of baseline CVD; (iii) a comparison of 3P-MACE in the ITT dataset stratified by a series of baseline characteristics ; (iv) a comparison between exendin or human GLP-1RA versus BI; (v) a re-match of groups excluding patients who initiated exendin-based GLP-1RA. For all analyses, we used Cox regression to estimate HRs and tested statistical significance at the 0.05 level. The E-value was calculated to assess the extent of confounding in attributing causality to the observational association between treatment and outcomes , 28.The statistical package R v4.0.1 and the python packages lifelines v0.21.0 and scipy v1.4.1 were used.The initial population of citizens of the Veneto Region was composed of 5,242,201 individuals. According to a validated algorithm, 330,193 could be identified as being affected by diabetes , 28,247 of whom had started a GLP-1RA or BI between 2014 and 2018. After excluding those treated with any insulin  or GLP-1RA in the previous year, we identified 22,187 patients newly treated with GLP-1RA (n = 4738) or BI . Figure The median duration of follow-up was 24 IQR: 12–36) months with the ITT approach, 17 months (IQR: 12–30) with the AT approach. In the primary analysis (ITT), we recorded 532 3P-MACE, 199 among patients who initiated GLP-1RA (25.8 events/1000 patient year) and 333 among patients who initiated BI (43.4 events/1000 patient year). The corresponding HR was 0.59  in favor of the GLP-1RA group , but was statistically significant and clinically relevant also for MI and HHF. Of note, when we limited the observation to the period effectively covered by drug prescription (AT approach), outcomes were even more in favor of GLP-1RA versus BI, with a 71% lower relative hazard of death. This result supports the biological plausibility of our findings, because limiting the observation to the period when patients were actually taking GLP-1RA or BI should indeed uncover the true difference between the two approaches.In the literature, we found a striking scarcity of studies reporting a similar observational comparison between GLP-1RA and BI. In the analysis of a large U.S. insurance claims database (2011–2015), use of GLP-1RA was associated with lower and use of BI was associated with higher rates of cardiovascular events when compared to use of DPP-4i, but no direct comparison was performed between injectable strategies . In a smWe acknowledge that typical biases occur when retrospectively comparing the outcomes associated with use of insulin versus those associated with use of non-insulin therapies. This is because patients with T2D who receive insulin are supposedly frailer than those using non-insulin drugs . UsuallyAs noted above, the lack of key clinical variables remains the major limitation of our study, which amplifies the ever-holding confounding by indication in observational research. Yet, the HR for all-cause mortality associated with GLP-1RA versus BI was close to the point where confounding is generally considered unlikely to be a major issue . ConsistOur study has other notable strengths as it represents, so far, the largest and most contemporary comparison of cardiovascular outcomes of patients treated with GLP-1RA versus BI under routine care, using state-of-the-art methods for comparative observational research.Patients with T2D who initiated a GLP-1RA experienced far better cardiovascular outcomes than did matched patients who initiated a BI in the same Region and healthcare system. Although part of this striking difference could be due to residual confounding by indication, our findings support the consensus on the use of GLP-1RA as the first injectable therapy for the management of T2D. In parallel, we acknowledge that the GLP-1RA/BI association is a valuable option in the management of T2D, either as fixed-ratio or loose combination. It can be helpful in the subsequent steps of therapeutic intensification and to simplify regimens as an alternative to basal-bolus insulin , 41. TheAdditional file 1: Table S1. Claims-based definition of study variables. Each variable was defined as the presence of at least one of the claims-based indicators in the corresponding row. Medication names have been internally mapped 1:1 to ATC codes, exemptions from copayment to regional exemption codes. Table S2. Available clinical-laboratory variables for the subset of patients in the matched cohorts. Standardized mean differences (SMD) are shown along with p-values. Table S3. Adverse events. Results of Cox regression on adverse events, and corresponding event rates . HR are calculated for SGLT2i vs. DPP4i initiators. GUTI, genitourinary tract infections. Figure S1. Between group balance before and after matching. The plot shows the absolute standardized mean difference between the group of patients who initiated GLP-1RA or BI for each variable before and after propensity score matching. Matching achieved a robust balance between groups, as evident from a differences < 10% for all variables. Figure S2. Distribution of propensity scores. Distribution of propensity scores (PS) is shown for initiators of GLP-1RA and basal insulin in the cohorts before and after propensity score matching (PSM). Note that the sum of the area under curves is always equal to 1.0, despite different sample size before and after PSM. Figure S3. Comparative cardiovascular outcomes by type of GLP-1RA. The Forest plot shows hazard ratios (HR) and 95% confidence intervals (CI) of all study endpoints in patients who initiated BI versus those who initiated a human- or exendin-based GLP-1RA. The numbers of patients in each subgroup are reported. In addition to p values of the HR in each subgroup, the interaction term (group × CVD) p value (p int.) is also reported.


Lignin is a natural light-coloured ultraviolet (UV) absorber; however, conventional extraction processes usually darken its colour and could be detrimental to its UV-shielding ability. In this study, a sustainable way of fabricating lignin–cellulose nanocrystals (L-CNCs) from hemp hurd is proposed. A homogeneous morphology of the hemp particles was achieved by ball milling, and L-CNCs with high aspect ratio were obtained through mild acid hydrolysis on the ball-milled particles. The L-CNCs were used as filler in polyvinyl alcohol (PVA) film, which produced a light-coloured nanocomposite film with high UV-shielding ability and enhanced tensile properties: the absorption of UV at wavelength of 400 nm and transparency in the visible-light region at wavelength of 550 nm was 116 times and 70% higher than that of pure PVA, respectively. In addition to these advantages, the nanocomposite film showed a water vapour transmission property comparable with commercial food package film, indicating potential applications. Though traditional metal oxidant materials  and guaiacyl (G) phenolic groups responsible for UV-shielding properties of lignin . Lignin In this paper, lignin is categorised as purified or native lignin ,9. The pCurrently, there are only a few reports available on native lignin–cellulose nanocrystals (L-CNCs) preparation, mainly based on acid hydrolysis; however, one of the key drawbacks is the low aspect ratio (around 11) of L-CNCs in the reported methods , limitinThe L-CNCs with high aspect ratio is difficult to achieve due to the crosslink barrier of lignin. The layers of lignin and the crosslinks between cellulose and lignin are two main obstacles of breaking down the cellulose to nanocellulose . MechaniTherefore, this study aims to propose a novel method of using a ball milling integrated acid hydrolysis technique to achieve high aspect ratio L-CNCs (preserving the UV-shielding) as a sustainable alternative to the current available techniques. We have chosen hemp hurd, since it is a lignin-rich resource containing around 16–24 wt. % ,22 of liThe hemp hurd (200 g by weight) was dried at 60 °C for 12 h, followed by chopping into a coarse particle with a Fritsch Pulverisette 19 Universal Cutting Mill (sieve diameter of 200 µm). Polyvinyl alcohol solution (PVA) (5 wt. %) was purchased from Flew solution . Sulphuric acid (98 wt. %) was purchased from Chemsupply .Hemp hurd particle (50 g) was wet ball-milled with an Attritor mill  at an agitator speed of 280 rpm for 20 h, as reported before . In our Aqueous solution of 4 g hemp and 25 mL sulphuric acid (64 wt. %) were added into a round bottle flask to hydrolyse for 1, 3, 5, 7, and 10 h under 45 °C, as shown in The temperature was kept at 45 °C based on the optimised method of fabricating CNCs described in the literature . Though The prepared L-CNCs samples  were added to the PVA solution, producing five different mixtures with a weight ratio of 5 wt. %. L-CNCs7, L-CNCs10 were selected to perform further comparison with the addition ratio of 10 wt. % and 20 wt.%. The hemp particle/PVA sample at a ratio of 5 wt.% was prepared from our previous study [To measure the total lignin in hemp hurd, the extractives were first removed according to NREL/TP-510-42619  is likelw/w) H2SO4 for 2 h at 20 °C, followed by adding 575 mL DI water to dilute into 4% (w/w) H2SO4 and boiled for 4 h. The hydrolysis solution and sediment were separated by filtering and washing with hot water. The weight of dried sediment was reported as acid-insoluble lignin. Acid-soluble lignin was measured from the hydrolysis solution by UV–vis spectroscopy, where the absorbance was used to calculate the weight of acid-soluble lignin. The acid-soluble lignin was calculated by the equation from the NREL/TP-510-42618 [The lignin in hemp hurd included both acid-insoluble lignin and acid soluble lignin. The content of acid-insoluble lignin was determined according to TAPPI T Standard 222 . Dried s10-42618  and was The stability and dispersion of L-CNCs in an aqueous solution were evaluated by Zeta potential. The value of Zeta potential was measured on a Zetasizer Nano  at 25 °C. Three measurements were carried out for each sample, and the average was reported. The pH value of L-CNCs suspension was controlled at 7 by diluting the L-CNCs solution 1000 times. This is worth mentioning that the dynamic light scattering (DLS) size is usually used to measure round particles by using the Stokes–Einstein relationship . Since tThe qualitative analysis of sulphur content in the hemp particle and L-CNCs was analysed by the energy-dispersive X-ray spectroscopy (EDS) method using a scanning electron microscope (SEM) JEOL7800F . The sulphur content was representing the percentage of the respective sample.The yield of hemp particles and L-CNCs was measured by using the gravimetric analysis method . The weiTo understand the influence of acid hydrolysis on the aspect ratio of L-CNCs, the length, width, and height of L-CNCs were evaluated by AFM . The L-CNCs solution was dropped on the surface of a silicon wafer and dried in a fume hood overnight. The lengths and heights of L-CNCs were measured on 30 random crystals captured by AFM images. The profile drawn by the long axis of the particle represents the length of L-CNCs, and the height represent the maximum value along the long axis . The widThe morphology of L-CNCs was imaged using JEOL TEM LaB6 . It operated at an acceleration voltage of 200 kV and beam current of 108 μA. The L-CNCs samples were diluted with DI water 1000 times and dropped on a carbon grid. The carbon grid was dried under fume hood for 12 h prior to testing.−1 and a resolution of 4 cm−1 was used to obtain the spectra. Opus 5.5 software was used to perform the baseline correction of the results. Origin 2020  was used to normalise and find the peaks. The syringyl-to-guaiacyl ratio was calculated by the intensity of I1328/I1244 of L-CNCs, where I1328 and I1244 represented the peak intensity in 1328 cm−1 and 1244 cm−1, respectively [A Bruker Vertex spectrometer  with attenuated total reflectance Fourier-transform infrared (ATR-FTIR) was used to understand the chemical structure of the L-CNCs and L-CNCs/PVA films. A light beam with a wavelength in the range 600 to 4000 cmectively .UV-shielding performance of the L-CNCs solution was determined with a UV spectrophotometer . The spectra of the samples were obtained in a 1 cm quartz cuvette from 200 to 800 nm. To avoid the influence of lignin amount, the data were nominalised per total lignin mass of L-CNCs. Transparency and UV-shielding performance of the films were determined with a UV–VIS–NIR spectrophotometer . The spectra of the films were obtained at a scan rate of 600 nm/min from 200 to 800 nm.Xc is the crystalline fraction, Ac represents the crystalline area and Aa represents the amorphous area of the film. The crystalline area and amorphous area were deconvoluted through Gaussian function provided in Origin 2020.The crystallinity of the L-CNCs-PVA film was measured by XRD . The intensity in the 2θ range from 7° to 50° were collected in the operating condition at 30 kV and 40 mA. The crystallinity of the film was calculated by Equation (1) .(1)Xc=AA universal tensile tester machine  was used to measure Young’s modulus, maximum nominal stress, and elongation at break at 20 ± 2 °C temperature and 62  ±  2% of relative humidity. The film was cut into 30 × 10 mm according to ASTM D882–18 . The gauA moisture vapour transmission rate tester  was used to obtain the water vapour permeability (WVP) of L-CNCs/PVA composite films. According to TAPPI T 464 , the meap-value ≤ 0.05 indicates that the difference in data sets was statistically significant, and p-value > 0.05 indicates the difference in data sets was not statistically significant.As required, either one-way or two-way ANOVA tests were conducted to understand the difference among samples through IBM SPSS. The acid-soluble and insoluble lignin content (wt. %) of hemp and L-CNCs, along with the zeta potential, are shown in Though the changes in the amount of acid-insoluble lignin showed fluctuations, overall, it represented an increasing trend. The increase in lignin content was probably related to the loss of cellulose and hemicellulose in the hydrolysis process. The amorphous range of cellulose and hemicellulose were dissolved in the sulphuric acid . This coThe zeta potential was also used to quantitatively characterise the electrostatic stability of L-CNCs. High positive or negative values of zeta potential indicate adequate electrical double layer repulsion, which can avoid possible aggregation. The sulphur content (wt.%) of the samples is presented in There was no significant difference in zeta potential values among L-CNCs3, L-CNCs5 and L-CNCs7. The L-CNCs10 showed the highest negative zeta potential indicating the best stability of the L-CNCs samples; however, it lost the highest amount of acid-soluble lignin (3.4%) and showed the highest amount of acid-insoluble lignin (13.2%) among the other L-CNCs. Compared to other L-CNC samples, the hemp particle sample was not treated with acid. The acid was only applied in the measurement process which probably resulted in comparatively lower amount of acid soluble lignin than other samples. Overall, the total lignin amount in the samples was lower than that reported in the literature ,22. ThisThe AFM images and height measurements of L-CNCs after subsequent hydrolysis with acid  are shown in The changes in length (L) and height (H) were found under different hydrolysis times, as shown in After depolymerisation of the lignin–carbohydrate complex, cellulose was exposed, resulting in a reduction in length. The length/height aspect ratio (L/H) of L-CNCs7 was the highest (~9 times) among hemp particle and other L-CNCs. The acid hydrolysis treatment from 0 to 7 h resulted in elongated L-CNCs with a higher aspect ratio. With an increased hydrolysis treatment time, the length of L-CNCs further decreased. The higher aspect ratio of L-CNCs would be a benefit for the properties of the composite when reinforced with the polymer. While the aspect ratio of fabricated L-CNCs  has been reported between 9.2 and 11.5 , the L/H−1 represent the OH- stretching of intramolecular and intermolecular hydrogen bonds, phenolic groups, phenols from cellulose and lignin [−1, 2912 cm−1, 1244 cm−1, and 846 cm−1 represented C-H stretching of L-CNCs [−1 was observed, and this was assigned to the C=O stretch of alpha, beta-unsaturated ketones [−1 represented the aromatic units [−1 was related to CH2 symmetric bending in lignin [−1 represented CH3 stretching from cellulose and lignin [−1 and 1050 cm−1 represented the S=O stretching [−1, 1087 cm−1, and 1165 cm−1 were due to C-O stretching and assigned to the cellulose and hemicellulose. A drop in peak intensity at 1024 cm−1 was observed in the L-CNCs3 sample, which indicated the low cellulose content. This sudden drop of cellulose content could be related to the formation of CNCs. As observed in AFM image, the CNCs was started forming after 3 h acid hydrolysis. The peaks at 1328 cm−1 and 1244 cm−1 were assigned to the syringyl (s) and guaiacyl (G) [1328/I1244 [The chemical change of L-CNCs was analysed by FTIR as shown in d lignin . The peaf L-CNCs . Compare ketones . The peaic units  in the hn lignin . The band lignin . The pearetching  of L-CNCacyl (G) , respectacyl (G) . Lignin acyl (G) . Therefo28/I1244 .−1 peak. Thus, this leads to the enhancement of S/G ratio and UV-shielding properties. The enhanced in S/G ratio was further confirmed by the enhanced UV-shielding properties of L-CNCs, as shown in the following UV–vis result. In addition, the results suggest that the UV-shielding properties of L-CNCs fabricated in this study is higher than softwood (S/G ratio of 0.053 for pine) or grass (S/G ratio 0.433 for switchgrass) and less than hardwood  [The calculated S/G ratio of hemp particle and L-CNCs are presented in alyptus) .−1 is due to the intermolecular and intramolecular hydrogen bond [−1, which was identical to the result from Haque et al. [−1. With the hydrolysis procedure, the absorption peak of L-CNCs shifted from 3325 cm−1 to 3275 cm−1, as shown in −1 was attributed to the residual acetyl groups in PVA [The hemp particle and L-CNCs were incorporated into PVA as a functional filler. The chemical bonding between particles and PVA were analysed by FTIR, as shown in gen bond . The hydgen bond ,59. PVA e et al. . The mais in PVA . The resThe UV–vis absorption spectra of hemp particle and L-CNCs are shown in The digital images of hemp particle and L-CNCs are shown in The hydrolysis influence on the UV-shielding performance was analysed by adding different L-CNCs samples with the same ratio (5 wt. %) in the PVA matrix. The UV-shielding performance and digital image of the prepared films are shown in The effect of L-CNCs content on the UV-shielding performance of the composite films is shown in UV-shielding properties of some lignin composite materials reported in the literature are shown in The influence of L-CNCs on mechanical properties, including Young’s modulus, maximum nominal stress, and elongation at break of PVA film, are shown in The L-CNCs7 showed a higher aspect ratio of 85.13 compared to those of L-CNCs3 (16.5), as proved by AFM results , thus leTo investigate the influence of filler amount, different contents of L-CNCs7 (highest aspect ratio) and L-CNCs10  were also added into PVA with 10 wt. % and 20 wt. %. Young’s modulus, maximum nominal stress, and elongation at break of those PVA composite films are shown in Water vapour permeability has been considered an important factor for packaging application, as it can influence the food shelf-life. One of the advantages of PVA is its low water vapour permeability, while adding filler normally influences its permeability. From the literature , the bar−12 g·cm/(cm2·s·Pa) [Normally, adding fillers could increase the pores of the film, leading to water molecule transfer through the film with less resistance. However, there were no statistical differences found between pure PVA and any of the samples. This could be related to two factors; one is the interaction between L-CNCs and PVA that restricted the passage of water molecules. The water molecules usually move through the amorphous region to permeate from one side to another. The less amorphous region also reduces the diffusion path, which means fewer water molecules pass through. Looking at food packaging materials such as HDPE (high-density polyethene), the water vapour permeability of HDPE has been reported at 1.741–3.482 × 10m2·s·Pa) , which iThis study demonstrated an innovative fabricating method for high aspect ratio native lignin-cellulose nanocrystals (L-CNCs) by ball milling assisted acid hydrolysis treatment. The proposed fabrication method is more environmentally friendly compared to the current methods, as it was developed using the mild concentration of sulphuric acid (57.25%) at a low temperature (45 °C). Although some acid-soluble lignin was lost through the acid hydrolysis treatment of hemp, the sulfonated L-CNCs showed higher UV absorption than those of hemp particle. Though the colour of L-CNCs was darker than hemp particle, light-coloured UV-shielding nanocomposite film was achieved by the casting of L-CNCs (with different hydrolysis times from 1 to 10 h) with PVA. Among all L-CNCs/PVA films at 5 wt. %, the L-CNCs7/PVA (hydrolysed for 7 h) was highly transparent (94.16% transmission at the wavelength of 550 nm) and showed good UV-shielding properties (73.46% absorption at the wavelength of 280 nm) due to the UV absorption of L-CNCs. At the filler addition of 10 wt. %, the L-CNCs7 (with the highest aspect ratio of 85.13) showed higher transparency and better UV-shielding properties compared to the L-CNCs10, having the smallest size among all L-CNCs. The evaluated tensile properties and water vapour transmission proved the potential application of developed L-CNCs nanocomposite film in food packaging.